Congenital Anomalies in American Crocodile (Crocodylus acutus, Cuvier, 1807) Embryos from a Farm Breeder in Colombia

Simple Summary Congenital defects have been described in almost every vertebrate group. In crocodiles, teratology alterations have been described in captive animals (pets, zoos, farms) such as Crocodylus niloticus and Gavialis gangeticus. The present study aimed to characterize congenital malformations of C. acutus from a farm in Lomas de Matunilla, Ballestas, Bolívar, Colombia. The analyzed eggs presented macroscopic malformations, with 42 different types of anomalies observed. Limb and tail malformations (29%) were the most common changes observed. Abstract The American crocodile (Crocodylus acutus, Cuvier, 1807) (Class Reptilia, Family Crocodylidae) is a crocodile species inhabiting the Neotropics. Congenital defects have been described in almost every vertebrate group. In crocodiles, teratology alterations have been described in captive animals (pets, zoos, farms) such as Crocodylus niloticus or Gavialis gangeticus. The present study aimed to characterize congenital malformations of C. acutus from a farm in Lomas de Matunilla, Ballestas, Bolívar, Colombia. A total of 550 unhatched eggs were examined after embryo death. A total of 61 embryos presented malformations, with 42 different types of anomalies observed. Limb and tail malformations (29%) were the most common malformations observed. Several malformations, such as cephalothoracopagus, thoracopagus, sternopagus, xiphopagus twins, campylorrachis scoliosa, and acrania, were documented in crocodiles for the first time. Research in teratology enhances our understanding of crocodile biology. It plays a role in their conservation and management, thus helping to ensure the long-term viability of these species in their natural habitats.


Introduction
American crocodile (Crocodylus acutus) (Class Reptilia, Family Crocodylidae) is a species of crocodile that inhabits the Neotropics like Belize, Colombia, Costa Rica, Cuba, the Dominican Republic, Ecuador, El Salvador, Guatemala, Haiti, Honduras, Jamaica, Mexico, Nicaragua, Panama, Peru, the United States (Florida), and Venezuela [1,2].According to the International Union for Conservation of Nature (IUCN)-Red List, this species Vet.Sci.2024, 11, 317 2 of 16 is considered Vulnerable [3].Its habitat consists mainly of coastal areas [4].C. acutus breed in late fall or early winter, and in February or March, the females lay 30 to 70 eggs in nests of sand, mud, and dead vegetation along the water bodies [5].The incubation period is about 75-80 days.The temperature at which the eggs are incubated (dependent on the environment) can influence the sex of the hatchlings, a phenomenon known as temperature-dependent sex determination (TSD).Warm temperatures during incubation tend to produce females, while cooler temperatures produce males [6].Alteration of the temperatures can alter the embryos [7].
Like other species, C. acutus faces various threats [3,8].Expanding cities and towns into coastal and wetland areas leads to the loss of crucial nesting and basking sites.The conversion of wetlands and coastal regions into agricultural lands destroys crocodile habitats.Agricultural runoff, industrial waste, and urban pollution degrade the quality of water bodies where these animals live.Dam construction and water diversion for agriculture and urban use can alter the natural flow of rivers and streams [9].In addition, illegal hunting exists due to the demand for crocodile skins, leather products, and meat [10].Climate change leads to rising sea levels inundating nesting sites, and temperature changes can skew sex ratios, impacting population dynamics [11].With the rise of human-crocodile conflicts, these animals sometimes prey on livestock or come to attack humans, leading to retaliatory killings [12].
Congenital defects have been described in almost every vertebrate group [13][14][15], but in wild animals, the descriptions of these anomalies are scarce, and the information is virtually non-existent [16].They are more commonly observed in captive animals [17].Most of the individuals who carry these malformations die before birth.If they survive, most malformations are only detected during a post-mortem exam [18].Teratogens, agents, or factors that can disturb the normal development of an embryo can include environmental agents like drugs, infectious diseases (e.g., viruses, bacteria, and fungi), radiation, temperature, pollution (e.g., heavy metals, and pesticides), or certain maternal conditions [13,18,19].
In reptiles, a wide variety of congenital anomalies has been reported in several species [20].For example, Martín-del-Campo et al. (2021) [21] showed the presence of malformation in three species of sea turtles associated with different environmental factors such as the incubation temperature, humidity, and the status of feeding areas [21].Other studies in South American pit vipers (Bothrops jararaca) and South American rattlesnakes (Crotalus durissus) linked the malformation to the presence of chemical compounds such as pesticides and herbicides [16].
In crocodiles, teratology alterations have been described in captive animals (pets, zoos, farms) in species such as Crocodylus niloticus, Gavialis gangeticus, Osteolaemus tetraspis, Alligator mississippiensis, C. porosus, C. johnsoni, C. palustris, or C. moreletii [22][23][24].Not all malformations can be considered as being from congenital origin, with some resulting from the exposure of embryos to environmental stressors [18].Pollution is a major concern, particularly chemical contaminants such as pesticides, heavy metals, and endocrine-disrupting compounds that can accumulate in aquatic ecosystems [25].These substances can interfere with normal developmental processes, leading to physical abnormalities.Habitat degradation due to urbanization, agriculture, and industrial activities can also play a role.Destruction of nesting sites and changes in water quality can create stressful conditions that increase the likelihood of malformations.Climate change and associated effects, like temperature fluctuations and altered precipitation patterns, can also disrupt embryonic development since crocodile sex determination and growth rates are temperature-dependent [22,26].
The present study aimed to characterize congenital malformations observed in American crocodiles (Crocodylus acutus) from a farm in Colombia.

Materials and Methods
Eggs of the American crocodile (Crocodylus acutus Cuvier, 1807) from a farm located in Lomas de Matunilla, Ballestas, Bolívar, Colombia (10°10′06″ N 75°28′46″ W) (Figure 1), were analyzed during Colombia's dry season (December to March) in 2023.From 61 nests across 15 lakes, 1773 eggs were laid from 61 females.The eggs were incubated and hatched from 25 April 2023 to 31 May 2023.Of the 1773 eggs, 1242 were incubated, with 48 eggs broken and 470 infertile eggs.Table 1 shows the information regarding nests, lakes, offspring, collection date, incubated eggs, laid eggs, broken eggs, infertile eggs, collected eggs, and dead embryos.From 61 nests across 15 lakes, 1773 eggs were laid from 61 females.The eggs were incubated and hatched from 25 April 2023 to 31 May 2023.Of the 1773 eggs, 1242 were incubated, with 48 eggs broken and 470 infertile eggs.Table 1 shows the information regarding nests, lakes, offspring, collection date, incubated eggs, laid eggs, broken eggs, infertile eggs, collected eggs, and dead embryos.All the eggs that did not hatch in the expected period (75-80 days) were analyzed.After embryo death, stillborn crocodiles were removed from the eggs and measured, systematically examined, and photographed with a scale.All the gross malformations were recorded in an Excel file and then categorized by different anatomical regions into skin, spine, head (excluding eye and nose), nose, limbs, tail, and other malformations.

Results
From the 1773 eggs laid, 550 eggs with dead embryos were analyzed from those 61 embryos that presented malformations.Overall, 95.7% (n = 57) presented more than one malformation.Forty-two different types of anomalies were observed (Table 2).

Skin Malformations
Ephitheliogenesis imperfecta (a localized region of abnormal skin) was observed in five animals, two of which had incomplete scale formation (Figure 3).Alterations of scale color were leucism (n = 2), where the scales were white crocodiles with dark markings and regional depigmentation to white (n = 3) (Figure 4).

Skin Malformations
Ephitheliogenesis imperfecta (a localized region of abnormal skin) was observed in five animals, two of which had incomplete scale formation (Figure 3).Alterations of scale color were leucism (n = 2), where the scales were white crocodiles with dark markings and regional depigmentation to white (n = 3) (Figure 4).

Spine Malformations
The main spine malformations were observed in fourteen animals with scoliosis (lateral curvature of the spinal column), three with lordosis (ventrodorsal deviation, curvature of the spinal column with a ventral convexity) (Figure 5), two with kyphosis (Increased dorsal convexity in the curvature of the spinal column as viewed from the side) (Figure 6), and one with campylorrachis scoliosa (curvature of the spine a lack of vertebrae and spinal cord caudal to the thoracic region) (Figure 7).

Spine Malformations
The main spine malformations were observed in fourteen animals with scoliosis (lateral curvature of the spinal column), three with lordosis (ventrodorsal deviation, curvature of the spinal column with a ventral convexity) (Figure 5), two with kyphosis (Increased dorsal convexity in the curvature of the spinal column as viewed from the side) (Figure 6), and one with campylorrachis scoliosa (curvature of the spine a lack of vertebrae and spinal cord caudal to the thoracic region) (Figure 7).

Spine Malformations
The main spine malformations were observed in fourteen animals with scoliosis (lateral curvature of the spinal column), three with lordosis (ventrodorsal deviation, curvature of the spinal column with a ventral convexity) (Figure 5), two with kyphosis (Increased dorsal convexity in the curvature of the spinal column as viewed from the side) (Figure 6), and one with campylorrachis scoliosa (curvature of the spine a lack of vertebrae and spinal cord caudal to the thoracic region) (Figure 7).
The main spine malformations were observed in fourteen animals with scoliosis (lateral curvature of the spinal column), three with lordosis (ventrodorsal deviation, curvature of the spinal column with a ventral convexity) (Figure 5), two with kyphosis (Increased dorsal convexity in the curvature of the spinal column as viewed from the side) (Figure 6), and one with campylorrachis scoliosa (curvature of the spine a lack of vertebrae and spinal cord caudal to the thoracic region) (Figure 7).

Head
The main malformations observed in the head were one anencephaly/prosencephalic hypoplasia (absence of the cranial region of the head, with the brain absent or reduced), twelve laterognathia (mandible pointing up and jaw pointing down), three meningoencephalocete (herniation of the brain and meninges through a cranial opening covered by skin), one brachygnathia (short lower mandible), four microcephaly (small head), three maxillary

Head
The main malformations observed in the head were one anencephaly/prosencephalic hypoplasia (absence of the cranial region of the head, with the brain absent or reduced), twelve laterognathia (mandible pointing up and jaw pointing down), three meningoencephalocete (herniation of the brain and meninges through a cranial opening covered by skin), one brachygnathia (short lower mandible), four microcephaly (small head), three maxillary micrognathia (short upper jaw), one external hydrocephalus (abnormal accumulation of cerebrospinal fluid within the ventricles and/or subarachnoid spaces, increase in head circum-

Head
The main malformations observed in the head were one anencephaly/prosencephalic hypoplasia (absence of the cranial region of the head, with the brain absent or reduced), twelve laterognathia (mandible pointing up and jaw pointing down), three meningoencephalocete (herniation of the brain and meninges through a cranial opening covered by skin), one brachygnathia (short lower mandible), four microcephaly (small head), three maxillary micrognathia (short upper jaw), one external hydrocephalus (abnormal accumulation of cerebrospinal fluid within the ventricles and/or subarachnoid spaces, increase in head circumference, combined with enlarged subarachnoid spaces), seven maxillary macrognathia (longer low jaw), three acrania (absence skull), one acephaslostomia, one maxilla agnathia (absent upper jaw), and one brachycephalic skull (shape of a skull shorter than average in its species) (Figures 8-13).

Other Malformations
In Achondroplasia (dwarfism), the long bones are abnormally short, although the trunk is of normal length and the abdomen is large (round)-the size of normal individuals.The head is moderately enlarged and flattened.This malformation was observed in four individuals (Figure 16).Four cases of twins were observed.Cephalothoracopagus (Figure 17) were symmetrical monozygotic twin crocodiles that conjoined from the head down to the Alterations of the tail observed were curled tail (curved into nearly a full circle) (n = 17), kinked tail (localized undulation(s) of the tail) (n = 18), acaudia (absent tail) (n = 16), brachyuri (n = 3), and bent tail (n = 2) (Figure 15).skull, ankylodactyly of the two digits fused in the left posterior member ( yellow), congenital cataract in both eyes, tubercles on top of the skull ( red), and epitheliogenesis imperfecta on the palpebra Crocodylus acutus embryo.((A)-Lateral body view, (B)-Cranial view, (C)-ventrodoral head view, (D)-lateral head view, (E)-ventrodorsal view).Scale bar = 5 cm.

Other Malformations
In Achondroplasia (dwarfism), the long bones are abnormally short, although the trunk is of normal length and the abdomen is large (round)-the size of normal individuals.The head is moderately enlarged and flattened.This malformation was observed in four individuals (Figure 16).Four cases of twins were observed.Cephalothoracopagus (Figure 17) were symmetrical monozygotic twin crocodiles that conjoined from the head down to the

Other Malformations
In Achondroplasia (dwarfism), the long bones are abnormally short, although the trunk is of normal length and the abdomen is large (round)-the size of normal individuals.The head is moderately enlarged and flattened.This malformation was observed in four individuals (Figure 16).Four cases of twins were observed.Cephalothoracopagus (Figure 17) were symmetrical monozygotic twin crocodiles that conjoined from the head down to the thorax with one head, one face, one neck, a single thorax, two abdomen, two umbilical cords, eight limbs, and two tails.There were three monozygotic symmetrical twins (identical twins) (Figure 18) and one thoracopagus, sternopagus, and xiphopagus twin (Twins attached by the thorax, sternum, and xiphoid process).We also observed one case of a distended celomic cavity, one case of Ectopia cordis (externalized heart), one case of gastroschisis (externalization of the internal organs abdomen), and 47 cases of yolk sac retention.
eight limbs, and two tails.There were three monozygotic symmetrical twins (identical twins) (Figure 18) and one thoracopagus, sternopagus, and xiphopagus twin (Twins attached by the thorax, sternum, and xiphoid process).We also observed one case of a distended celomic cavity, one case of Ectopia cordis (externalized heart), one case of gastroschisis (externalization of the internal organs abdomen), and 47 cases of yolk sac retention.thorax with one head, one face, one neck, a single thorax, two abdomen, two umbilical cords, eight limbs, and two tails.There were three monozygotic symmetrical twins (identical twins) (Figure 18) and one thoracopagus, sternopagus, and xiphopagus twin (Twins attached by the thorax, sternum, and xiphoid process).We also observed one case of a distended celomic cavity, one case of Ectopia cordis (externalized heart), one case of gastroschisis (externalization of the internal organs abdomen), and 47 cases of yolk sac retention.thorax with one head, one face, one neck, a single thorax, two abdomen, two umbilical cords, eight limbs, and two tails.There were three monozygotic symmetrical twins (identical twins) (Figure 18) and one thoracopagus, sternopagus, and xiphopagus twin (Twins attached by the thorax, sternum, and xiphoid process).We also observed one case of a distended celomic cavity, one case of Ectopia cordis (externalized heart), one case of gastroschisis (externalization of the internal organs abdomen), and 47 cases of yolk sac retention.

Discussion
In this work, the authors describe the congenital deformities in American crocodiles (Crocodylus acutus).To the the authors' knowledge, the present study is the first of this kind.
According to studies performed [27], crocodiles have a low prevalence of deformities even in captivity.In Gavialis gangeticus, deformities were detected in 6% of the 1061 hatchlings examined [28].
In this study, all the animals were born in captivity and died during gestation, except for one individual.This animal was born without a tail; it would be difficult for it to survive in the wild since it could not swim to the surface [24], but it is more likely to survive in captivity.Limbs and tail malformations (29%) were the most common changes observed, followed by head (25%).These results are similar to what has been described by other authors [6,24,29].Almost every congenital malformation observed in this study has been reported in other species of crocodiles [23,24,26].In the cases described were observed malformations such as cephalothoracopagus, thoracopagus, sternopagus, xiphopagus twins, campylorrachis scoliosa, acephaslostomia, and acrania, that were not described in crocodiles to the moment.
Malformations in wild animals are rarely described [22,27].These congenital malformations can be associated with many factors, such as hatchlings from very young and very old females, genetic causes, malnutrition of the parents, defective incubation, and carcinogenic agents [30].
For example, Alligator mississippiensis from Lake Apopka (USA) embryos presented a large number of malformations associated with a chemical spill of sulfuric acid, dicofol, organochlorine pesticides, and Polychlorinated biphenyls (PCBs) [29,31].South American pit vipers (Bothrops jararaca) and South American rattlesnakes (Crotalus durissus) malformations have been linked to the increased use of pesticides and herbicides in the agriculture fields near their habitat [16].Benzo(a)pyrene and dibenzo(a)anthracene, polychlorinated biphenyls (PCBs), and heavy metals (As, Cu, Cd, Hg, Pb) were related to the development of malformations in the semi-aquatic turtles such as Chelydra serpentina and Chrysemy spicta from the John Heinz National Wildlife Refuge (USA) [32].
There is the possibility that genes can be associated with these malformations if both parents are carriers.In gharial populations from Nepal, a 'blind' gene has been suspected to occur [28].In spite of our study, we did not confirm the etiology of the congenital malformations; a combination of many factors, such as the females' age, inbreeding, incubation errors, and chemical pollutants in the water (the farms' water originates in very polluted rivers in Columbia) should be considered.Although no samples were collected from the water in the farm lakes, it is very possible that pesticides like Organochlorine and heavy metals are present in such water since these compounds have been detected in other rivers in Colombia [29].
Although other studies have reported the occurrence of malformations in crocodiles and other reptiles, there is still a large gap concerning the classification of these malformations and defining the frequency with which they occur.Crocodiles are ectothermic carnivores that live in aquatic environments and may act as sentinel species [33,34].In the future, more detailed studies should be performed to find the potential role of genetic and environmental factors in the occurrence of the reported malformations in these species.

Conclusions
This study describes congenital deformities in American crocodiles (Crocodylus acutus).It is important to note that research in teratology not only enhances our understanding of the biology of crocodiles but also plays a role in their conservation and management, helping to ensure the long-term viability of these species in their natural habitats.Identifying the causes of developmental abnormalities can contribute to conservation efforts by addressing potential threats and improving management strategies.Although other studies have reported the occurrence of malformations in crocodiles, there is still a large gap concerning the classification of these malformations and the frequency at which they occur.Examining abnormal development can also offer insights into the genetic diversity of crocodile populations and potential genetic stressors.

Figure 1 .
Figure 1.Colombia map with the location of the American crocodile (Crocodylus acutus Cuvier, 1807) farm.

Figure 1 .
Figure 1.Colombia map with the location of the American crocodile (Crocodylus acutus Cuvier, 1807) farm.

Table 2 .
Malformations in American crocodile (Crocodylus acutus) from a farm in Colombia.